CELL STORAGE-02-Freezing and Thawing Protocol for Suspension Cell Lines v1

In our institute, cell lines are stored after freezing procedure that provides for a constant temperature lowering by 1 degree/minute. This procedure is useful to minimizes ice crystals formation during freezing process, this allow to reduce the cells damage and, in turn increasing cells viability after thawing. After storage at -80°C or in liquid nitrogen (in relation to subsequent applications or the expected freezing time) cells are thawed rapidly in a water bath at 37°C and the cryopreserving-medium is immediately remove by gentle centrifugation in order to reduce as soon as possible, the DMSO toxic effect. Currently, in our institute, the freezing/thawing procedure is validated for the following cell lines: PC-12, Rat adrenal phaeochromocytoma, small irregularly shaped cells morphology, suspension growth mode. After freezing/thawing procedure, cells quality must be assessed by cell morphology evaluation (using Optical Microscopy technique) and time required to reach confluence in the T25 cm2 culture flask, which must not exceed the times indicated in the Table 1. After quality test cells must be resuspended according to specific protocols (for details see Table 2). List of published work by our institute that using this procedure Cosentino M, Marino F, Rasini E, Legnaro M, Bombelli R, Luini A, Pacchetti B. Improved solubility and increased biological activity of NeoSol™RCL40, a novel Red Clover Isoflavone Aglycones extract preparation. Biomed Pharmacother. 2019 Mar;111:91-98. doi: 10.1016/j.biopha.2018.12.065. Epub 2018 Dec 19. PMID: 30579257.